FACTORS INFLUENCING SUCCESSFUL AGROBACTERIUMMEDIATED GENETIC TRANSFORMATION OF MUSKMELON

Document Type : Research articles.

Authors

1 Department of Genetics, Faculty of Agriculture, Fayoum University, Egypt

2 Department of Genetics and Plant Breeding, Faculty of Horticultural Sciences, Szent István University, Budapest, Hungary

Abstract

The development of a robust Agrobacterium-mediated
transformation protocol for muskmelon (Cucumis melo L.)
requires the identification and optimization of the factors affecting
DNA delivery and plant regeneration. We have used pieces of
mature cotyledon from cultivar Hales Best Jumbo and the
Agrobacterium tumefaciens strain LBA4404, to investigate and
optimize regeneration and transformation. Agrobacterium strain
harboring the binary vectors which contains phosphino-thricin
acetyl transferase gene (bar) as selectable marker for herbicide
resistance (glufosinat ammonium) and β-glucuronidase gene
(uidA) as reporter. Factors that produced differences in DNA
delivery and regeneration included bacterial concentration, length
of exposure, the time allowed for co-cultivation and antibiotic
concentration (claforan, cefotaximum). These factors combined as
a whole led to successful transformation. Bacterial concentration,
time of exposure of the explants to the bacteria, co-cultivation
period and antibiotic concentration were found to affect
regeneration and transformation efficiency. Analysis of these
parameters allowed the development of an optimized protocol for
Agrobacterium-mediated transformation of Cucumis melo cv.
Hales Best Jumbo cotyledon explants. We fully describe a
protocol that allowed efficient DNA delivery and gave rise stable
transgenic muskmelon plants. Selectable marker gene expression
and reporter gene assay demonstrated that transgenes were
integrated into the muskmelon genome. Genetic transformation of
calli and regenerated plantlets was confirmed by histochemical β-
glucuronidase assays.

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